1. Field of the Invention
The present invention generally relates to particles composed of a nonconducting core coated with a very thin metallic layer, and to methods of using these particles for sensing a chemical or biological analyte. More particularly, the invention relates to such particles having defined maximum absorption or scattering wavelengths, and, optionally, having one or more biomolecules conjugated to the metallic layer.
2. Description of Related Art
It has long been observed that an enormous enhancement of Raman scattering intensities is possible from many biologically significant organic molecules when they are adsorbed onto roughened silver electrodes or in a solution of aggregating colloid (Fleischmann, M. et al. J. Chem. Soc. Commun. 80 (1973); Duff, D. G., et al. Langmuir 9:2301 (1993)). This effect, known as surface enhanced Raman scattering (SERS), can yield a Raman spectrum as much as a million times stronger than the spectrum of the same molecule in solution. While this approach has been popular with Raman spectroscopy using visible excitation, SERS enhancement becomes almost a requirement when a near infrared excitation source is used, as in FT-Raman spectroscopy. Although infrared excitation eliminates sample fluorescence, it also results in marked decrease in sensitivity, further motivating the need for a sensitization method. Current methods being used for SERS enhancement of near infrared FT-Raman spectroscopy are frequently plagued by difficult substrate preparation, poor reproducibility, sensitivity to contamination, or limited suitability for in vivo use.
The SERS effect is primarily related to the field strength near the surface of the substrate upon illumination, whether the substrate is a roughened metal surface or an aggregate of metallic nanoparticles. The strongest field enhancement is obtainable at the plasmon resonance of the metal substrate or particle. It is for this reason that gold colloid (plasmon resonance=520 nm) is such an efficient SERS enhancer under visible Raman excitation (typically with an argon ion laser at 514 nm). This resonance coincides with the absorption maximum of hemoglobin (Gordy, E. et al. J. Biol. Chem. 227:285-299 (1957)), however, which significantly restricts the use of visible excitation Raman spectroscopy on biological systems.
The idea of exploiting SERS in biosensing applications has been pursued using other strategies for quite some time. Previous workers have used SERS to measure binding between biological molecules of mutual affinity, including antibody-antigen interactions (Rohr, T. E., et al. Anal. Biochem. 182:388-398 (1989)). The approach in that study included the use of an avidin-coated silver film as substrate and dye-antibody conjugates to optimally enhance the SERS effect. Although that method was used in a successful sandwich immunoassay, the use of a microscopic silver substrate and the necessity for conjugation of the biomolecules with specific (carcinogenic) chromophores for resonance Raman detection severely limits the adaptability of that approach.
U.S. Pat. No. 5,567,628 (Tarcha et al.) describes an immunoassay method for performing surface enhanced Raman spectroscopy. Various substrates are described, including solid particles of gold or silver. U.S. Pat. No. 5,869,346 (Xiaoming et al) describes an apparatus and method for measuring surface-sensitized Raman scattering by an antigen-antibody complex adsorbed to solid gold, silver or copper particles.
Optical glucose monitoring is one example of an extremely important and active field of research. The goal of this research is to provide a noninvasive method of monitoring and more optimally managing diabetes, a disease that affects millions of people worldwide. A variety of approaches are currently being pursued, including near- and mid-infrared spectroscopy, photoacoustic spectroscopy, polarimetry, diffuse light scattering, and Raman spectroscopy (Waynant, R. W., et al. IEEE-LEOS Newsletter 12:3-6 (1998)). In comparison to the other approaches in use, Raman spectroscopy with near infrared excitation offers the unique ability to discriminate between spectra from different analytes even when signals are small. Raman spectroscopy is the only all-optical technique currently under consideration in which the entire spectral signature of a chemical species can be obtained. The spectral signature is not obscured by water, and the significant penetration depth achieved with near-IR excitation ( greater than 1 mm) facilitates a variety of in vivo monitoring approaches. Raman spectroscopic measurements of glucose in human blood serum and ocular aqueous humor (using both conventional Raman and stimulated Raman gain spectroscopy) have also been reported (Wicksted, J. P., et al. App. Spectroscopy 49:987-993 (1995); and U.S. Pat. No. 5,243,983 issued to Tarr et al.). Since near infrared excitation results in a dramatic decrease in sensitivity relative to visible Raman excitation, the most outstanding current limitation to Raman-based glucose monitoring is the lack of sensitivity. This results in the necessity of long data collection times and multivariate analysis techniques for signal extraction.
The use of gold colloid in biological applications began in 1971, when Faulk and Taylor invented the immunogold staining procedure. Since that time, the labeling of targeting molecules, especially proteins, with gold nanoparticles has revolutionized the visualization of cellular or tissue components by electron microscopy (M. A. Hayat, ed. Colloidal Gold: Principles, Methods and Applications Academic Press, San Diego, Calif. 1989). The optical and electron beam contrast qualities of gold colloid have provided excellent detection qualities for such techniques as immunoblotting, flow cytometry and hybridization assays. Conjugation protocols exist for the labeling of a broad range of biomolecules with gold colloid, such as protein A, avidin, streptavidin, glucose oxidase, horseradish peroxidase and IgG (M. A. Kerr et al., eds. Immunochemistry Labfax BIOS Scientific Publishers, Ltd., Oxford, U.K. 1994).
Metal nanoshells are a new type of xe2x80x9cnanoparticlexe2x80x9d composed of a non-conducting, semiconductor or dielectric core coated with an ultrathin metallic layer. As more fully described in co-pending U.S. patent application Ser. No. 09/038,377, metal nanoshells manifest physical properties that are truly unique. For example, it has been discovered that metal nanoshells possess attractive optical properties similar to metal colloids, i.e., a strong optical absorption and an extremely large and fast third-order nonlinear optical (NLO) polarizability associated with their plasmon resonance. At resonance, dilute solutions of conventional gold colloid possess some of the strongest electronic NLO susceptibilities of any known substance. (Hache, F. et al. App. Phys. 47:347-357 (1988)) However, unlike simple metal colloids, the plasmon resonance frequency of metal nanoshells depends on the relative size of the nanoparticle core and the thickness of the metallic shell (Neeves, A. E. et al. J. Opt. Soc. Am. B6:787 (1989); and Kreibig, U. et al. Optical Properties of Metal Clusters, Springer, N.Y. (1995)). The relative thickness or depth of each particle""s constituent layers determines the wavelength of its absorption. Hence, by adjusting the relative core and shell thicknesses, and choice of materials, metal nanoshells can be fabricated that will absorb or scatter light at any wavelength across much of the ultraviolet, visible and infrared range of the electromagnetic spectrum. Whether the particle functions as an absorber or a scatterer of incident radiation depends on the ratio of the particle diameter to the wavelength of the incident light. What is highly desirable in the biomedical field are better, more sensitive devices and methods for performing in vivo sensing of chemical or biological analytes. Also desired are easier, more rapid and more sensitive methods and reagents for conducting in vitro assays for analytes such as autoantibodies, antiviral or antibacterial antibodies, serum protein antigens, cytokines, hormones, drugs, and the like.
Methods of in vitro and in vivo sensing of chemical or biochemical analytes employing SERS enhanced Raman spectroscopy are provided. Special metal coated particles (xe2x80x9cmetal nanoshellsxe2x80x9d), with or without conjugated biomolecules, and having diameters ranging from a few nanometers up to about 5 microns and defined wavelength absorbance or scattering maxima across the ultraviolet to infrared range of the electromagnetic spectrum are employed in the methods and compositions of the present invention.
One aspect of the invention provides a composition useful for biosensing applications. In certain embodiments, the composition comprises a plurality of particles and a support. In some embodiments the support comprises a medium such as a hydrogel matrix. In other embodiments the support comprises a substrate on which the particles are arrayed. Each particle comprises a non-conducting core having an independently defined radius and a metal shell adhering to the core and having an independently defined thickness. The terms xe2x80x9cindependently defined radiusxe2x80x9d and xe2x80x9cindependently defined thicknessxe2x80x9d mean that the desired thickness of each of the shell and core can be chosen and formed without dictating or requiring a certain thickness of the other. Each particle has a defined core radius:shell thickness ratio, a defined absorbance or scattering maximum wavelength (when measured in the same medium) in the ultraviolet to infrared range of the electromagnetic spectrum. The particle also has a surface capable of inducing surface enhanced Raman scattering, and, optionally one or more biomolecules conjugated to the particle surface. In some embodiments, a reporter molecule is conjugated to the shell or to the biomolecule. A reporter molecule could be an enzyme, a dye molecule, a Raman sensitive chemical, or the like. In some embodiments the conjugated biomolecule or the shell surface itself has an affinity for the analyte, causing at least some analyte molecules to adsorb or closely associate with the surface of the particle (e.g., localize within about 50-100 nm of the particle""s surface, and preferably within about 10-20 nm of the surface). In some embodiments the support, or a portion of the support, has an affinity for the analyte sufficient to cause it to similarly localize near the surface of the particles. In certain preferred embodiments of the composition, the particles and the medium are in the form of a matrix such as a hydrogel that is permeable to an analyte of interest. Another aspect of the invention provides methods of making an optically tuned nanoshell especially for use in biosensing applications. The term xe2x80x9coptically tuned nanoshellxe2x80x9d means that the particle has been fabricated in such a way that it has a predetermined or defined shell thickness, a defined core thickness and core radius:shell thickness ratio, and that the wavelength at which the particle significantly, or preferably substantially maximally absorbs or scatters light is a desired, preselected value. For example, the selected wavelength of significant absorbance may correspond to an absorbance maximum (peak), or it may correspond to any strongly absorbed wavelength that falls on the xe2x80x9cshoulderxe2x80x9d of an absorbance peak, or the selected wavelength may fall within a strongly absorbing plateau region of the particle""s absorbance spectral curve. It should be understood that the term xe2x80x9cmaximum absorbancexe2x80x9d also includes this meaning, whenever the context applies. The particle""s wavelength of significant absorbance may be chosen to substantially match a certain laser peak wavelength. A preferred embodiment of this method includes selecting the desired wavelength of light (xcexmax) at which light of a selected wavelength will be significantly absorbed or scattered by the particle. A non-conducting core of radius Rc is formed, and then a metal shell is grown or deposited onto the core, the final shell having a thickness Ts. This method also includes controlling the ratio of Rc:Ts such that the wavelength of light maximally absorbed or scattered by the particle is approximately xcexmax in the UV to infrared range of the electromagnetic spectrum. In some embodiments, one or more analyte specific molecules, which may be a biomolecule such as an antibody, an antigen or an enzyme are conjugated to the shell. In certain embodiments a reporter molecule is instead or additionally conjugated to the shell or to the analyte specific molecule. The selected xcexmax preferably corresponds to the desired wavelength of the incident light that is to be employed when the nanoshells are used in a particular biosensing application.
Yet another aspect of the invention provides an in vitro method of assaying a biological analyte in a sample (e.g., blood, serum, or other body fluid). For example, the biological analyte could be a chemical or a biomolecule, such as proteins (e.g., antibodies, antigens and enzymes), peptides, oligonucleotides and polysaccharides, or a conjugate thereof.
According to certain embodiments, the in vitro assay method includes selecting one or more optically tuned nanoshells with an absorption or scattering maximum wavelength that substantially matches the wavelength of a desired source of electromagnetic radiation. In some embodiments the chosen nanoshells include one or more conjugated biomolecules. The method also includes associating the nanoshells with one or more molecules of the desired analyte contained in the sample such that an analyte/nanoshell complex is formed. In certain embodiments the method includes associating the nanoshells with a reporter molecule, in which case a reporter/analyte/nanoshell complex is formed. Either complex is capable of producing a Raman signal upon irradiation by the selected source. Preferably the source is in the near-IR range of the electromagnetic spectrum. The method further includes irradiating the complex with incident electromagnetic radiation at the predetermined wavelength so that surface enhanced Raman scattering is induced. A Raman scattering signal from the complex is detected and the signal is correlated to the presence and/or amount of the analyte in the biological sample. In preferred embodiments a SERS signal is also detected in the near-infrared range. A major advantage of the nanoshell biosensing technology of the present invention is that the need for indicator enzymes in many types of bioassays is obviated, which allows analysis of biological samples with little or no prior purification steps. Because a strong SERS signal from molecules right at the surface of the nanoshells can be obtained, other xe2x80x9ccontaminatingxe2x80x9d molecules in the unpurified or bulk sample, such as serum or whole blood, do not interfere with spectral response measurements of the molecule of interest.
In a further aspect of the present invention, a kit is provided for conducting nanoshell-based immunosorbent assays. These assays may be of the sandwich-type, direct- or indirect-types, analogous to the respective conventional immunosorbant assays. In one embodiment, the kit includes a quantity of a first antibody-nanoshell conjugate, and, optionally, a quantity of a control antigen having affinity for binding to the first antibody. This kit may also optionally include a quantity of a secondary antibody that has affinity for binding to an antigen-first antibody-nanoshell conjugate. In some embodiments a reporter molecule is bound to the second antibody. The nanoshells in the kit comprise a non-conducting core having an independently defined radius, a metal shell adhering to said core and having an independently defined thickness a defined core radius:shell thickness ratio, a defined absorbance wavelength maximum in the ultraviolet to infrared range, and a surface capable of inducing surface enhanced Raman scattering.
A further aspect of the present invention provides an in vivo method of monitoring a biological analyte. A preferred embodiment of this method comprises introducing a quantity of optically tuned metal nanoshell particles into a subject at a desired biosensing site in the body. In certain embodiments the site is internally accessible to an analyte of interest and is accessible to externally applied electromagnetic radiation. In other embodiments the site is accessible to the analyte and is also irradiated via an internally placed light source, as in a totally implantable system, for example. The particles are optically tuned such that the wavelength of light that is maximally absorbed or scattered by the particles substantially matches the wavelength of light emitted from a predetermined source of electromagnetic radiation in the ultraviolet to infrared range. For example, the average peak wavelength of a group of particles could be within about 10-15 nm of the 1064 nm wavelength of a Nd:Yag laser. Preferred embodiments of the method include selecting a source of electromagnetic radiation emitting light at a wavelength that matches said maximally absorbed or scattered wavelength. In certain embodiments the particles have an affinity for the analyte, and in some embodiments include a reporter molecule, which in certain embodiments contains a Raman active functional group. The method also includes externally applying radiation to the particles and any analyte molecules associated with the particles so that a SERS signal is produced. The method includes evaluating the signal and correlating a signal evaluation with the presence and/or amount of the analyte at the biosensing site.
Certain embodiments of the in vivo method of monitoring a biological analyte includes fabricating a quantity of optically tuned particles such that the wavelength of light that is maximally absorbed or scattered by said particles substantially matches the wavelength of light emitted from a predetermined source of ultraviolet-infrared electromagnetic radiation.
In accordance with still another aspect of the invention, a particle for biosensing applications is provided. The particle, also referred to as a metal nanoshell, comprises a non-conducting or dielectric core having an independently defined radius, a metal shell closely adhering to the core and having an independently defined thickness, and a defined core radius to shell thickness ratio. The particle also has a defined or predetermined wavelength absorbance or scattering maximum in the 300 nm to 20 xcexcm range of the electromagnetic spectrum. In some embodiments the defined wavelength absorbance or scattering maximum is in the near-infrared range. In some embodiments, the maximum absorbance wavelength of the particle is set at about 800-1,300 nm or about 1,600-1,850 nm. In certain preferred embodiments, the particle has a wavelength maximum that substantially matches the peak wavelength of a given source of electromagnetic radiation and has a surface that is capable of inducing surface enhanced Raman scattering. In certain embodiments the metal shell has a surface with an affinity for associating analyte molecules.
In some embodiments of the particle of the invention, the particle has one or more analyte binding molecules conjugated to the metal shell surface. In certain embodiments the analyte binding molecule is a biomolecule, such as a protein, polypeptide, oligonucleotide or polysaccharide. In some embodiments the analyte binding molecule is a mixture of species of biomolecules conjugated to the shell. In certain embodiments the biomolecule is glucose oxidase and the analyte is glucose, and in certain other embodiments the biomolecule is an antibody and the analyte is a target antigen for the antibody. In certain preferred embodiments the shell comprises gold or silver, and the core comprises a material such as silicon dioxide, gold sulfide, titanium dioxide, polymethyl methacrylate (PMMA), polystyrene or a macromolecule such as a dendrimer. A preferred embodiment of the particle of the invention, especially suited for use in biosensing, has a gold shell and a silicon dioxide core. Other preferred nanoshells have a silver shell and a silicon dioxide core. The diameter of some of these particles is up to about 5 xcexcm, with the core diameter being about 1 nm to nearly 5 xcexcm, and the shell thickness being about 1-100 nm. In certain of the more preferred embodiments, the core is between 1 nm and 2 xcexcm in diameter and the shell is less than 40 nm thick. In this embodiment, the shell is linked to the core through a linker molecule, and the particle has a wavelength of maximum absorbance or scattering between 300 nm and 20 xcexcm. In some embodiments the particle is about 210 nm in diameter, has a core radius of about 100 nm, a shell thickness of about 10 nm, a core radius:shell thickness of about 10:1, and a maximum absorbance wavelength (xcexmax) of about 1064 (SDxc2x110 nm), substantially matching the 1064 nm (peak) Nd:YAG source as used in a FT-Raman laser spectrometer. Preferred embodiments of the particle of the invention have a gold shell or silver shell. Preferred embodiments of the particle have a core that comprises silicon dioxide, gold sulfide, titanium dioxide, polymethyl methacrylate (PMMA), polystyrene and macromolecules such as dendrimers. In another aspect of the invention, a chemical sensing device comprising certain of the above-described particles is provided. The chemical sensing device may be, for example, an all-optical sensor employing suitably designed nanoshells and SERS spectroscopy to detect and quantify a drug or a plasma protein such as a particular anti-viral or anti-bacterial antibody or a given cytokine.